Many genetic variations (including germ-line and somatic mutations) are important markers for hereditary abnormality, disease progression and therapeutic efficacy. Molecular diagnostic assays based on various technologies have been or are being developed to detect single nucleotide polymorphisms (SNPs). One of the widely adopted methods is allele specific polymerase chain reaction (AS-PCR) in which allele specific primers are designed to amplify variant specific targets based on the selective extension by polymerase according to the 3′ matching between the primer and its template. Specifically, PCR amplification is only sufficiently effective where there are no or very few mismatches between the primer and its template at or near the 3′ end of the primer, whereas PCR amplification is not detectable when the number of mismatches at or near the 3′ end of the primer is sufficient to disrupt effective binding of the primer to the template.
The sensitivity and specificity of such methods significantly depend on the differential PCR efficiencies between the templates containing the SNPs of interest and non-targeted templates containing other sequences, including other alleles in the case of germ-line mutations and wild-type (or other mutations) in the case of somatic mutations. When the difference in PCR efficiency between the targeted and non-targeted templates is insufficient, there may be detectable amplification on the non-targeted template (non-specific signals), and the non-specific amplification signals (e.g., Ct or signal strength), albeit less efficient, can be too close to the specific signals (e.g., Ct or signal strength) to fully separate low level of specific targets from the non-specific targets. In such cases, it is difficult to establish assay cut-off to achieve both high level sensitivity and specificity. The technical requirement of fully differentiated PCR efficiencies is particularly critical in areas where very low mutant contents need to be detected from the samples, such as many onocology-associated somatic mutations.
The BRAF gene, an example of a gene having an onocology-associated somatic mutation, encodes a protein belonging to the raf/mil family of serine/threonine protein kinases (namely, serine/threonine-protein kinase B-raf). B-raf plays a role in regulating the MAPK (mitogen-activated protein kinase) signaling pathway, which affects cell division, cell differentiation, and secretion. Germ-line mutations in the BRAF gene are associated with cardiofaciocutaneous syndrome, which is characterized by heart defects, a distinctive facial appearance, and mental retardation. Mutations in the BRAF gene are also associated with various types of cancers, including adenocarcinoma of the lung, colorectal cancer, malignant melanoma, non-Hodgkin's lymphoma, non-small cell lung carcinoma, and thyroid carcinoma.
Mutation of thymine at nucleotide position 1796 to adenine has been detected in lung cancers and head and neck cancers (U.S. Pat. No. 7,378,233; see U.S. Pat. No. 7,442,507 for T1799A). Detection of T1796A in exon 15 of the BRAF gene reportedly enables a malignant papillary thyroid neoplasm to be distinguished from a benign thyroid sample (U.S. Pat. No. 7,378,233) and also enables distinction of HNPCC tumors from sporadic colorectal tumors (Int'l Pat. App. Pub. No. WO 2005/071109). Detection of T1799A reportedly indicates the presence of metastatic melanoma (U.S. Pat. App. No. 2006/0246476, now U.S. Pat. No. 7,442,507).
Most mutations in the BRAF gene associated with cancers occur at amino acid position 600, which is located in the activation domain. Amino acid position 600 also has been referred to as amino acid position 599 in the literature. Mutation of valine (V) at amino acid position 600 to glutamic acid (E) (see, e.g., U.S. Pat. App. Pub. No. 2007/0020657, Davies, et al., Nature 417: 949-954 (2002), in which it is designated V599E, and Kimura, et al., Cancer Res. 63: 1454-1457 (2003)), lysine (K), or aspartic acid (D) accounts for more than 90% of all mutations in the BRAF gene. The presence of a colorectal neoplasm reportedly can be determined by detecting a point mutation in an exfoliated epithelial marker, such as BRAF, along with one or more fecal occult blood markers (see U.S. Pat. App. Pub. No. 2011/0236916). Analysis of BRAF mutations, along with microsatellite stability, reportedly enables prognosis of survival rates in patients with cancer as well as classification of severity of cancer in patients (see Int'l Pat. App. Pub. No. WO 2007/009013 and U.S. Pat. App. Pub. No. 2009/0181371). See, e.g., U.S. Pat. App. Pub. No. 2011/0269124 and Int'l Pat. App. Pub. No. WO 2011/019704 for detection of BRAF mutations generally. Mutation in codon 599 of exon 15 of BRAF reportedly enables the detection of malignant melanoma (see U.S. Pat. App. Pub. No. 2007/0087350; see, also, Int'l Pat. App. Pub. Nos. WO 2010/097020, WO 2005/027710, WO 2005/059171, and WO 2005/066346). The use of real-time polymerase chain reaction (PCR) clamping based on peptide nucleic acid (PNA) to detect mutations in codon 600 in BRAF is described in Int'l Pat. App. Pub. No. WO 2011/093606, whereas the use of allele-specific real-time quantitative PCR (AS-QPCR) using locked nucleic acid primers and beacon detectable oligonucleotides to detect V600E mutations in BRAF is described in Int'l Pat. App. Pub. No. WO 2011/104694 and the use of fluorescent quantitative PCR to detect mutations in the BRAF gene is described in Int'l Pat. App. Pub. No. WO 2011/103770. Liquid chips for detecting a V600E mutation in the BRAF gene are described in Int'l Pat. App. Pub. No. WO 2011/131146. Therefore, the ability to detect single nucleotide polymorphisms (SNPs) that lead to mutations of V600N599 would provide important information about the diagnosis and prognosis of cancer.
In addition to providing information about cancer diagnosis and prognosis, the ability to detect SNPs that lead to mutations of V600/V599 also would provide important information about the therapeutic efficacy of drugs targeting the MAPK pathway. Detection of a mutation in codon 600 of BRAF, such as V600E by amplification of a polynucleotide sequence comprising V600E, reportedly enables the determination of the sensitivity of cancer cells to a B-raf kinase inhibitor (see U.S. Pat. App. Pub. Nos. 2010/0173294 and 2011/0212991). Detection of homozygous/heterozygous V600E or V600D genotype or any genotype characterized by BRAF gain-of-function phenotype reportedly enables evaluation of sensitivity of malignant/neoplastic cells to ERK1/ERK2/MEK inhibitors (see U.S. Pat. App. Pub. No. 2011/0158944; see also Int'l Pat. App. Pub. No. WO 2009/073513). The detection of a mutation in BRAF, such as V600E, reportedly enables the generation of a personalized report for treatment of a patient with colon cancer with cetuximab or panitumumab (see U.S. Pat. App. Pub. No. 2011/0230360). Shinozaki, et al., Clin. Cancer Res. 13: 2068-2074 (2007), discloses the analysis of circulating B-RAF DNA mutations in serum for monitoring melanoma patients receiving biochemotherapy. Methods of optimizing treatment of cancer based on BRAF mutations, as well as other methods, are described in Int'l Pat. App. Pub. No. WO 2011/106298.
Existing methods for detecting BRAF mutations, such as sequencing, pyrosequencing, array, shifted termination assay (STA), polymerase chain reaction (PCR) followed by dual-priming oligonucleotide (DPO) PCR, and real-time PCR utilizing either allele-specific primers or allele-specific detectable oligonucleotide are accompanied by various disadvantages (see, e.g., Benlloch, et al., J. Mol. Diagn. 8: 540-543 (2006), for comparison of automatic sequencing and real-time chemistry methodology in the detection of BRAF V600E mutation in colorectal cancer; see, e.g., Hay, et al., Arch. Pathol. Lab. Med. 131: 1361-1367 (2007), for melting curve analysis of PCR products used to identify BRAF mutations in melanocytic lesions and papillary thyroid carcinoma samples; see, e.g., Jarry, et al., Mol. Cell. Detectable oligonucleotides 18: 349-352 (2004), for real-time allele-specific amplification in the detection of BRAF V600E; see, e.g., Sapio, et al., Eur. J. Endocrinol. 154: 341-348 (2006), for use of mutant allele-specific PCR amplification (MASA) to detect BRAF mutation in thyroid papillary carcinoma; and, see, e.g., Turner, et al., J. Cutan. Pathol. 32: 334-339 (2005), for use of the ligase detection reaction to detect BRAF V600E in melanocytic lesions). Sequencing and pyrosequencing methods are limited by their sensitivity, with the lowest detectable mutant content around 10-20% (% mutant over total background). Other methods, such as real-time PCR utilizing allele-specific detectable oligonucleotides, STA, array, and PCR/DPO are also limited by their sensitivity. Ideally, a sensitivity of 1% or better is desired.
Existing methods that lack sufficient specificity, such as real-time PCR, cannot differentiate between specific types of mutations, such as V600E and V600K. Even though V600E accounts for 90% of all mutations found at this amino acid position, other amino acid substitutions with clinical significant have been found in various cancers, sometimes with high prevalence, such as V600K in melanoma. Thus, the ability to differentiate between specific types of mutations is becoming increasingly important.
Assay workflow and automation are critical aspects of any diagnostic method. For certain technologies, such as sequencing, existing assay procedures are long and complicated. Other technologies, such as PCR/DPO (dual priming oligo) and array-based methods, may require extensive sample handling post-PCR, which is prone to amplicon contamination. In certain cases, additional steps have to be included in order to achieve differentiated detection of multiple mutations.
The present disclosure seeks to overcome some of the disadvantages attendant currently available methods of detecting germ-line and somatic mutations, especially those associated with hereditary abnormalities, disease progression and therapeutic efficacy. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein.